C    E 


3RARV    RePrmted  from  THE  BOTANICAL  GAZETTE,  42:  127-134,  August,  1906 

W/f/Uj  3A 


*&*  *>"*  (I    IAGRICI 

ON  THE  IMPORTANCE  OF  PHYSIOLOGICALLY 

BALANCED  SOLUTIONS  FOR  PLANTS.1  ^. 

I.    MARINE  PLANTS. 

W.    J.    V.    OSTERHOUT.  CALr 

RINGER  demonstrated  that  animal  tissues  live  longer  in  a  solut 
of  NaCl  to  which  a  small  amount  of  KC1  and  CaCl2  is  added  than 
in  a  solution  of  NaCl  alone.  Various  explanations  of  this  fact  were 
given  by  different  investigators,  all  of  whom,  however,  agreed  upon 
the  essential  point  that  KC1  and  CaCl2  are  essential  for  the  mainte- 
nance of  life. 

HOWELL  assumed  that  CaCl2  is  the  stimulus  for  the  heart  beat, 
while  NaCl  is  an  indifferent  substance,  necessary  only  for  the  mainte- 
nance of  osmotic  pressure.  Similarly  RINGER  concluded  that  Ca  is 
the  stimulus  for  the  systole,  while  K  is  necessary  for  the  diastole  of 
the  heart  beat. 

HERBST  made  experiments  on  the  influence  of  the  composition  of 
the  sea  water  on  sea  urchin  eggs,  eliminating  in  each  successive 
experiment  a  different  constituent  of  the  sea  water.  He  found  that 
the  eggs  would  not  develop  in  any  solution  which  did  not  contain 
all  the  salts  of  the  sea  water.  From  this  he  concluded  that  each  of 
the  salts  found  in  sea  water  is  necessary  for  the  development  of  the 
egg.  LOEB  called  this  view  in  question  as  the  result  of  his  experiments 
on  Fundulus.  He  found  that  this  marine  fish  cannot  live  in  a  pure 
NaCl  solution  of  the  same  osmotic  pressure  as  the  sea  water,  but  that 
it  can  live  indefinitely  in  a  mixture  of  NaCl,  KC1,  and  CaCl2,  in  the 
same  proportions  in  which  these  salts  are  contained  in  sea  water. 
The  fish  can  also  live  indefinitely  in  distilled  water.  This  proves 
that  it  does  not  need  any  of  the  three  salts  mentioned  for  the  mainte- 
nance of  its  life,  and  that  the  Ca  and  K  are  only  required  to  overcome 
the  poisonous  effects  which  would  be  produced  by  the  NaCl  if  it 
alone  were  present  in  the  solution  (at  the  above  mentioned  concen- 
tration). 

1  I  wish  here  to  express  my  sincere  thanks  to  Professor  LOEB,  who  kindly  placed 
the  facilities  of    his  laboratory  at  my  disposal  and  assisted  me  in  every  way    during 
these  investigations. 
127]  [Botanical  Gazette,  vol.  42 


128  BOTANICAL  GAZETTE  [AUGUST 

It  is  noteworthy  that  the  Ca  and  K,  which  are  added  to  inhibit 
the  toxic  effect  of  NaCl,  are  themselves  poisonous  at  the  concentra- 
tion at  which  they  are  here  employed. 

These  antagonistic  effects  of  Ca  and  K  toward  a  pure  NaCl  solu- 
tion were  illustrated  still  more  strikingly  in  experiments  on  the  egg 
of  Fundulus.  The  newly  fertilized  eggs  of  this  fish  develop  equally 
well  in  sea  water  and  in  distilled  water,  but  die  in  a  pure  m/2  NaCl 
solution  without  forming  an  embryo.  If,  however,  a  small  but  defi- 
nite amount  of  a  salt  with  a  bivalent  kation,  even  of  such  poisonous 
salts  as  BaCl2,  ZnSO4,  and  Pb(CH3-COO)2,  is  added,  the  eggs  will 
produce  embryos.  From  these  and  similar  observations  LOEB  was 
led  to  formulate  his  conception  of  the  necessity  of  physiologically 
balanced  salt  solutions,  in  which  are  inhibited  or  counteracted  the 
toxic  effects  which  each  constituent  would  have  if  it  alone  were 
present  in  the  solution. 

The  blood,  the  sea  water,  and  to  a  large  extent  RINGER'S  solution, 
are  such  physiologically  balanced  salt  solutions.  The  observations 
of  HERBST,  as  well  as  those  of  RINGER,  are  easily  explained  on  this 
basis.  The  fact  that  the  elimination  of  any  one  constituent  from 
the  sea  water  makes  the  solution  unfit  to  sustain  life  does  not  prove 
that  the  eliminated  substance  is  needed  by  the  animal  for  any  purpose 
other  than  to  counteract  the  poisonous  action  of  some  other  constit- 
uent of  the  solution. 

Botanists  have  not  thus  far  made  use  of  these  conclusions,  for  the 
obvious  reason  that  facts  similar  to  those  mentioned  above  have  not 
been  observed  in  plants.  I  have  recently  made  a  number  of  experi- 
ments which  show  that  there  exist  in  plants  phenomena  similar  to 
those  observed  by  LOEB  on  Fundulus  and  other  marine  animals. 

The  species  of  marine  plants  chosen  for  investigation  may  be 
divided  into  two  groups: 

Group  i  comprises  plants  which  can  live  a  long  time  in  distilled 
water.  It  includes  the  following:  BLUE-GREEN  ALGAE,  Lyngbya 
aestuarii;  GREEN  ALGAE,  Enteromorpha  Hopkirkii;  FLOWERING 
PLANTS,  Ruppia  maritima. 

Group  2  is  composed  of  plants  which  quickly  die  in  distilled 
water.  It  includes  the  following:  GREEN  ALGAE,  Enteromorpha 
intestinalis;  BROWN  ALGAE,  Ectocarpus  confervoides ;  RED  ALGAE, 


I9o6]  6 STERH OUT— BALANCED  SOLUTIONS  129 

Ptilota  filicina,  Pterosiphonia  bipinnata,  Iridaea  laminarioides, 
Sarcophyllis  pygmaea,  Nitophyllum  multilobum,  Porphyra  naiadum, 
Porphyra  perforata,  Gelidium  sp.,  Gymnogongrus  linearis,  Gigartina 
mammillosa.2 

If  plants  of  either  group  be  placed  in  a  solution  of  pure  sodium 
chlorid  (isotonic  with  sea  water),  they  die  in  a  short  timer- -This 
might  be  attributed  to  the  lack  of  certain  salts  which  are  necessary 
for  their  metabolism,  rather  than  to  the  toxicity  of  the  sodium  chlorid. 
In  the  case  of  the  plants  of  Group  i  there  can  be  no  doubt  on  this 
point,  for  these  plants  live  a  long  time  in  distilled  water.  If  we  add 
pure  sodium  chlorid  to  the  distilled  water  it  kills  them  in  a  very 
short  time.  An  inspection  of  the  tables  will  show  that  these  plants 
in  their  behavior  toward  sodium  chlorid  and  other  salts,  closely 
agree  with  those  of  Group  2,  which  can  live  but  a  short  time  in  dis- 
tilled water.  Sodium  chlorid  is  certainly  toxic  to  the  first  group, 
and  there  can  be  little  doubt  that  it  is  so  to  the  second  group  as 
well. 

The  plants  of  the  first  group  were  found  in  a  ditch  in  a  salt  marsh 
through  which  the  tide  ebbs  and  flows;  there  is  always  a  foot  or 
so  of  water  even  at  low  tide.  The  salt  content  of  the  water  fluctuates 
around  a  mean  of  approximately  2 . 3  per  cent. 

The  plants  of  the  second  group  were  collected  at  the  entrance  to 
San  Francisco  Bay,  where  the  salt  content  of  the  water  fluctuates 
about  a  mean  which  is  probably  not  far  from  2.7  per  cent.  The 
only  exceptions  are  Enteromorpha  intestinalis  and  Ectocarpus  con- 
jervoides,  which  came  from  wharves  in  the  bay,  where  the  mean  salt 
content  is  about  2.3  per  cent. 

All  the  plants  used  in  the  experiments  were  transferred  from  the 
sea  water  directly  to  distilled  water.  After  rinsing  in  this  they  were 
placed  in  glass  dishes,  each  containing  2oocc  of  the  solution  to  be 
tested.  The  dishes  were  then  covered  with  glass  plates  to  exclude 
dust  and  check  evaporation.  Only  a  small  amount  of  material  was 
placed  in  each  dish.  The  temperature  during  the  experiments  did 
not  vary  far  from  18°  C. 

Artificial  sea  water  was  prepared3  according  to  VAN  'T  HOFF'S 

2  The  determinations  were  kindly  made  by  Professor  SETCHELL. 

3  The  water  used  was  distilled  in  glass  only  and  the  first  part  of  the  distillate 
rejected.     The  purity  of  each  salt  was  carefully  tested  before  using. 


130  BOTANICAL  GAZETTE  [AUGUST 

formula4  as  follows:  iooocc  NaCl,  3^/8;  y8cc  MgCl2,  3^/8;  38CC 
MgSO4,  3^/8;  22CC  KC1,  3^/8;  iocc  CaCla,  3w/8.s 

This  closely  approximates  the  bay  water.  The  plants  thrive 
almost  as  well  in  it  as  in  sea  water,  especially  when  a  very  little 
NaHCO3  or  KHCO3  is  added  to  produce  a  neutral  or  faintly  alka- 
line reaction. 

A  series  of  solutions  was  tried,  beginning  with  pure  NaCl  3^/8 
and  adding  to  it  in  turn  MgCl2,  KC1,  and  CaCl2,  either  singly  or  in 
combination,  in  the  proportions  given  above.  These  salts  were  also 
used  in  pure  solutions  of  the  same  concentration  at  which  they  exist 
in  the  artificial  sea  water  described  above. 

It  should  be  said  that  little  difficulty  was  experienced  in  deter- 
mining the  death  point  with  sufficient  precision.  The  color  reactions 
and  the  microscopic  appearance  of  the  cells  allowed  this  to  be  done 
with  sufficient  accuracy,  so  that  the  results  were  not  in  doubt  on  this 
account. 

The  results  of  the  experiments  are  set  forth  in  the  tables.  The 
figures  represent  the  average  of  four  parallel  series  carried  on  simul- 
taneously. A  control  series  was  also  carried  on  in  which  each  solu- 
tion was  made  faintly  alkaline  by  the  addition  of  NaHCO3,  KHCO3, 
or  Ca(OH)2.  This  had  a  beneficial  effect  during  the  first  two  or 
three  days  of  the  experiment,  but  the  final  results  were  practically 
the  same  as  in  the  other  series. 

From  a  consideration  of  the  results  for  Group  i  we  may  draw 
the  following  conclusions. 

i.  The  plants  die  much  sooner  in  a  pure  sodium  chlorid  solution 
(isotonic  with  sea  water)  than  in  distilled  water.  The  poisonous 
effect  of  the  NaCl  largely  disappears  if  we  add  a  little  CaCl2  (iocc 
CaCl2  3^/8  to  iooocc  NaCl  3^/8) ;  in  this  mixture  the  plants  live 
nearly  as  long  is  in  distilled  water.  Addition  of  KC1  to  this  mix- 
ture enables  them  to  live  longer  than  in  distilled  water.  Further 
addition  of  MgCl2  and  MgSO4  enables  them  to  live  practically  as 
long  as  in  sea  water. 

4  VAN'T  HOFF,  J.  H.,  Physical  chemistry  in  the  service  of  the  sciences  101.  Univ. 
of  Chicago  Press,  1903. 

s  This  corresponds  approximately  to  the  proportion  of  Ca  in  the  sea  water  of 
the  bay. 


I9o6] 


OSTERHOUT— BALANCED  SOLUTIONS 


TABLE  I. 
DURATION  OF  LIFE  IN  DAYS. 


GROUP  i 

GROUP  2 

CULTURE  SOLUTION. 

Lyngbya 
aestuarii 

E  n  t  e  r  o- 
morpha 
Hopkirkii 

Ruppia 
maritima 

Ptilota 
filicina 

Pterosi- 
phonia  — 
bipinnata 

Tridaea 
Lominar- 
ioidcs 

Sea  water  (total  salts  2.7%) 

95 

150  + 

150  + 

II 

24^ 

24 

Artificial  sea  water: 

1000  c     NaCl       sm/S 

78  '     MgCl2 

38  '     MgS04       " 

90 

150  + 

150  + 

io| 

24^ 

23 

22    '      KC1 

10  '     CaCl2 

Distilled  water  

30 

80 

! 

,     2* 

Tap  water  

32  + 

36 

8s 

2| 

oi 

-  2 
IO 

NaCl      3^/8 

22 

O 

15 

0 

23 

Ij 

y  2 

4 

1000  cc  NaCl           "      ) 
10  "    CaCl2          "     J 

29 

23 

65 

4 

6 

5 

1000  "   NaCl           "     ) 

22    "     KC1                   "        £ 

35 

32 

88 

3^ 

10 

9 

10  "   CaCl2          "     ) 

1000  "   NaCl           "     ) 

78  "    MgCl2         "      [ 

29 

23 

45 

3 

6 

6 

10  "    CaCl2                 ) 

1000  "   NaCl           "     ) 

78  "   MgCl2         "     ( 

25 

13* 

30 

2 

4 

4 

22    "     KC1                   "        ) 

1000  "   NaCl           "     ) 

22    "     KC1                   "        ] 

23 

13* 

23 

I 

2 

5 

1000  "   NaCl           "      ) 
78  "    MgCl2         "      \ 

22| 

!3i 

25 

I* 

2 

2 

1000  "    Dist.  H2O          ) 
78  "    MgCl2         «     J 

* 

16} 

•9 

I 

2 

2* 

1000  "    Dist.  H2O          I 

38  "    MgS04        «      \ 

»7i 

13 

23 

I 

2 

2 

1000  "    Dist.  H2O         ) 

22    "     KC1                   "       J 

21 

•31 

56 

I 

.1 

s* 

1000  "   Dist.  H2O         ) 
10  "    CaCl2          "      \ 

26  + 

.4 

58 

a* 

5 

2 

132 


BOTANICAL  GAZETTE 


[AUGUST 


TABLE  II. 
DURATION  OF  LIFE  IN  DAYS.     GROUP  2. 


rt 

&^ 

«J 

a  rt 

el 

B 

5 

1 

1 

CULTURE  SOLUTION. 

Enteromo 
intestin 

&fc 

<rf«*H 

Sarcophy 
pygmac 

jl 

Porphyra 
naiadu 

Porph\Ta 
per  tor  a 

Gelidium 
sp. 

1 

o 

Gigartina 
mamm 

Sea  water  (total  salt  2  .  7  %.)  

240 

25 

II 

4* 

6 

21 

33  + 

ii 

II 

Artificial  sea  water  : 

1000  cc  NaCl      3/w/8; 

78  ||    MgCl,         ||     1 

22O 

2O 

7* 

4i 

6 

20 

33  + 

IO 

0} 

22    "      KC1                  "       ( 

10  "    CaCl2 

Distilled*  water  

•2 

1 

i5 

2i 

2) 

3* 

i| 

2* 

^ 

Tap  water  

IO 

4 

3f 

3f 

2$ 

4§ 

5* 

5l 

NaCl       3W/8 

§ 

7 

•j 

5| 

1000  cc  NaCl           "     ) 

22    "     KC1                  "        [ 

68 

8 

el 

^1 

cr 

Tit 

,,  _l_ 

Q 

6 

10  "    CaCl2          "     ) 

1000  "    Dist.  H2O         ) 

it 

4 

T& 

if 

•2 

^ 

4 

7 

•22    "      KC1                   "        )    " 

2.  The  pure  solution  of  each  of  the  salts  added  to  inhibit  the 
poisonous  effects  of  NaCl  is  itself  poisonous  at  the  concentration 
at  which  it  exists  after  its  addition,  since  the  plants  die  in  such  a  solu- 
tion much  sooner  than,  in  distilled  water.6  A  mixture  of  solutions 
which  are  individually  poisonous  produces  a  medium  in  which  the 
plants  live  indefinitely. 

That  the  plants  die  so  quickly  in  solutions  containing  a  single  salt 
might  be  attributed  to  the  fact  that  the  osmotic  pressure  of  some 
of  these  solutions  is  much  lower  than  that  of  sea  water.  This  sup- 
position is  disproved  by  the  fact  that  in  general  the  plants  live  longer 
in  tap  water  than  in  any  solution  containing  but  a  single  salt,  although 
the  tap  water  has  a  lower  osmotic  pressure  than  that  of  any  solu- 
tion used  in  the  experiments.  (The  plants  of  Group  i  live  longer 
in  distilled  water  also.  The  tap  water  is  to  be  regarded  as  a  physi- 

6  This  statement  does  not  apply  in  all  cases  to  CaCl2,  which  is  the  least  toxic  of 
the  salts  employed  and  for  some  forms  quite  harmless  in  dilute  solutions. 


1906]  OSTERH OUT— BALANCED  SOLUTIONS  133 

ologically  balanced  solution;    this  will  be   more    fully  discussed  in 
the  second  portion  of  the  paper.) 

3.  The  poisonous  effect  of  NaCl  is  inhibited  little  or  not  at  all 
by  KC1  or  MgCl2  added  singly. 

4.  The  combination   NaCl-f  KCl  +  CaCl2  is  superior  to  NaCl  + 
MgCl2  +  CaCl2,  but  the  latter  is  better  than  NaCl  +  MgC^^Cd. 

5.  These  effects  must  be  due  to  the  metal  ions,  since  the  anion 
is  in  nearly  all  cases  the  same. 

The  plants  of  Group  2  agree  with  those  of  Group  i  except  in  their 
behavior  toward  distilled  water. 

Essentially  similar  results  were  obtained  from  the  study  of  fresh 
water  algae  and  other  plants,  the  details  of  which  will  be  given  in 
the  second  part  of  this  paper. 

These  results  agree  in  striking  fashion  with  those  obtained  from 
the  study  of  marine7  and  freshwater  animals8. 

The  combination  NaCl  +  KCl  +  CaCl2  (in  the  same  proportions 
as  in  sea  water)  seems  to  be  quite  generally  beneficial  for  animals 
and  plants. 

We  may  in  conclusion  briefly  consider  the  effects  of  concentrated 
solutions.  A  series  of  experiments  were  made  on  Enter omorpha 
Hopkirkii  in  which  the  plants  were  placed  in  dishes  with  a  very  little 
sea  water.  This  quickly  evaporated,  so  that  the  plants  became 
covered  with  salt  crystals  in  24  to  48  hours.  In  this  condition  some 
of  them  remained  alive  for  about  150  days.  This  means  that  Entero- 
morpha  plants  which  remain  alive  only  15  days  in  3^/8  NaCl  solu- 
tion can  live  150  days  in  an  NaCl  solution  of  10  to  12  times  .higher 
concentration,  provided  the  other  salts  of  the  sea  water  are  present 
in  the  solution  (at  corresponding  concentration)  to  inhibit  the  toxic 
effect  of  NaCl.  Experiments  on  Lyngbya,  Ptilota,  and  Pterosiphonia 
gave  essentially  the  same  results. 

In  view  of  these  results,  and  others  of  a  similar  character  shortly 
to  be  published,  it  appears  certain  that  physiologically  balanced  salt 
solutions  have  the  same  fundamental  importance  for  plants  as  for 
animals. 

7  LOEB,  Pfliiger's  Archiv  107:252.  1905,  and  the  literature  there  cited. 

8  OSTWALD,  Pfluger's  Archiv  106:568.  1905.     Univ.  of  California  Publications, 
Physiology  2:163.  I9°5- 


134  BOTANICAL  GAZETTE  [AUGUST 

RESULTS. 

T.  Each  of  the  salts  of  the  sea  water  is  poisonous  where  it  alone 
is  present  in  solution. 

2.  In  a  mixture  of  these  salts  (in  the  proper  proportions)  the 
toxic  effects  are  mutually  counteracted.     The  mixture  so  formed  is 
a  physiologically  balanced  solution. 

3.  Such  physiologically  balanced  solutions  have  the  same  funda- 
mental importance  for  plants  as  for  animals. 

THE  UNIVERSITY  OF  CALIFORNIA, 
Berkeley. 


Reprinted  from  the  BOTANICAL  GAZETTE,  44:  259-272,  October  1907 


ON  THE  IMPORTANCE  OF  PHYSIOLOG- 
ICALLY BALANCED  SOLUTIONS^ 
FOR  PLANTS 

II.  FRESH-WATER  AND  TERRESTRIAL  PLANTS 
(WITH  SEVEN  FIGURES) 


W.  J.  V.  OSTERHOUT 


PRINTED  AT  THE  UNIVERSITY  OF  CHICAGO  PRESS 


ON    THE   IMPORTANCE    OF   PHYSIOLOGICALLY 
BALANCED   SOLUTIONS  FOR  PLANTS 

II.     FRESH-WATER  AND  TERRESTRIAL  PLANTS- 

W.   J.   V.    OSTERHOUT 

(WITH  SEVEN  FIGURES) 

If  the  facts  set  forth  in  the  first  part1  of  this  paper  prove  to  be  valid, 
not  for  marine  plants  only,  but  also  for  all  other  kinds,  we  cannot 
suppose  them  to  be  merely  the  result  of  adaptation  to  a  particular 
environment,  but  must  consider  them  to  be  the  direct  expression  of 
certain  fundamental  characteristics  of  living  matter.  In  order  that 
the  evidence  on  this  important  point  might  be  as  complete  as  possible, 
a  wide  range  of  material  was  studied.  It  includes  both  lower  and 
higher  algae,  liverworts,  Equisetaceae,  and  several  species  of  flower- 
ing plants,  embracing  among  the  latter  both  fresh-water  aquatics 
and  land  plants.  The  solutions  were  made  up  with  all  the  precautions 
regarding  distilled  water  and  purity  of  salts  described  in  the  first 
part  of  this  paper.  The  solutions  had  the  same  compositions  as 
there  described,  except  that  lower  concentrations  were  employed. 
A  control  series  was  always  made,  in  which  all  solutions  were  made 
faintly  alkaline.  The  material  was  always  rinsed  in  distilled  water 
before  being  placed  in  the  solutions.  The  plants  were  in  all  cases 
exposed  to  fairly  bright  light,  but  not  to  direct  sunlight.  The  tem- 
perature averaged  between  18°  and  2O°C.,  and  was  not  subject  to 
much  fluctuation. 

ALGAE 

The  most  extensive  series  of  experiments  on  algae  was  made 
with  Vaucheria  and  Spirogyra.  AfoimofVaucheriasessilis,  abun- 
dant in  running  water,  was  chosen  because  it  readily  gives  off  zoo- 
spores  when  brought  into  the  laboratory.  Tufts  of  this  material, 
washed  free  from  all  adhering  dirt,  were  placed  in  glass  dishes  and 
covered  with  tap  water.  Glass  slides  were  placed  upright  in  the 
dishes.  On  the  following  morning  numerous  zoospores  were  found 

1  BOTANICAL  GAZETTE  42: 127-134.  1906. 
259]  [Botanical  Gazette,  vol.  44 


260 


BOTANICAL  GAZETTE 


[OCTOBER 


O      O 


attached  to  each  slide  at  the  water  level.     As  many  as  fifty  to  a 

hundred  zoospores  were  commonly 
.         found  arranged  in  a  row  across  the 
"  slide,  so  that   subsequent  observa- 

tion was  an  easy  matter.  The  slides 
were  thoroughly  rinsed  in  distilled  water  and 
transferred  to  the  solutions.  The  solutions 
were  contained  in  glass  tumblers,  in  which 
the  slides  were  placed  in  an  upright  position, 
care  being  taken  to  have  the  zoospores  always 
at  the  same  depth  below  the  surface.  The 
volume  of  the  solution,  ioocc,  was  very  large 
compared  with  that  of  the  zoospores.  The 
tumblers  were  covered  with  glass  plates  to 
exclude  dust  and  hinder  evaporation.  Under 
these  circumstances  the  plants  thrive  excellently 
and  in  favorable  solutions  produce  normal 
mature  fruit.2 

•  The  average  results  of  six  series  of  experi- 
ments are  given  in  Table  III  and  illustrated  in 
fig.  i.  The  figure  shows  clearly  how  a  mixture 
of  two  poisonous  substances  may  produce  a 
solution  as  harmless  as  distilled  water. 

The  species  of  Spirogyra  employed  is  a  large 
one  of  the  majuscula  type.  The  material  was 
transferred  from  the  pond  directly  to  distilled 
water;  after  being  rinsed  in  this  it  was  placed 
in  covered  glass  dishes  containing  each  200 cc  of 
solution. 

It  will  be  seen  that  these  results  agree  in 
the  most  striking  way  with  those  already 
described  for  marine  plants. 

Further  experiments  were  made  with  a 
variety  of  other  algae,  including  a  species  of 
blue-green  alga  (Oscillatoria),  Chlamydomonas, 
Closterium  and  two  other  species  of  desmids, 

2  Cf.  OSTERHOUT,  Extreme  toxicity  of  sodium  chloride  and  its  prevention  by 
other  salts.     Jour.  Biol.  Chemistry  1:363.  1906. 


FIG.  i.  —  Growth  of 
zoospores  of  Vaucheria 
during  25  days  in  various 
w/ioo  solutions.  The 
quantities  are  stated  in 
cubic  centimeters,  the 
length  in  millimeters, 
and  the  gain  in  length 
in  per  cent,  i,  distilled 
water,  length  9.4,  gain 
5000.  2,  NaCl  1000 
+  CaCl2  10,  length  9.4, 
gain  5000.  3,  NaCl, 
length  0.18,  gain  o.  4, 
CaCl2,  length  0.18, 
gain  o. 


1907] 


OSTERHOUT— BALANCED  SOLUTIONS 


261 


a  diatom  (Navicula),  and  a  species  of  Oedogonium.  The  results 
agree  closely  with  those  given  below.  There  can  be  little  doubt, 
therefore,  that  the  algae  in  general,  both  fresh-water  and  marine, 
obey  the  same  law. 

TABLE  III.     ALGAE 

A  plus  sign  indicates  that  the  plants  were  alive  when  the  experiment  was  stopped. 
All  quantities  given  are  cubic  centimeters  of  3^/32  solutions. 


CULTURE  SOLUTION 


Dilute  sea  water  (total  salts  0.6  per  cent.)  40  + 

Dilute  artificial  sea  water: 
1000  NaCl 
78  MgCl2 

38  MgS04      40  + 

22  KC1 
10  CaCl2 

Distilled  water 40  + 

Tap  water 40  + 

Nad 
1000  NaCl 

10  CaCl2     J 

1000  NaCl      ) 

22  KC1         [ 40  + 

10  CaCl2     ) 

1000  NaCl      ) 

78  MgCl2    [• 40  + 

10  CaCl2     ) 

1000  NaCl 
78  MgCl2 
22-KC1 

1000  NaCl 

22    KC1 

1000  NaCl 

78    MgCla 

'"^Sfecu'-o 

1000  Dist.  H2O         „,/._..  Tj-pi 

22  KC1  =w/495  KU. 


DURATION  or  LIFE  IN  DAYS 


Vaucheria 


Spirogyra 


95  + 

95  + 
95  + 

95 

60 
65 
65 


95  + 


LIVERWORTS 


The  gemmae  of  Lunularia  furnish  ideal  material  for  studies  on 
the  effects  of  solutions.  They  can  be  obtained  at  any  time  of  year, 
they  grow  readily  when  floating  on  the  surfaces  of  solutions,  and 
are  fairly  uniform  in  behavior.  The  material  used  in  these  experi- 


262 


BOTANICAL  GAZETTE 


[OCTOBER 


ments  was  obtained  in  part  from  a  greenhouse  and  in  part  from  moist 
banks  of  earth  along  streams.  Material  from  different  sources  was 
never  mixed  together  in  any  series  of  experiments. 


o    G    o 

456 


FIG.  2. — Growth  of  gem- 
mae of  Lunularia  in  various 
3w/8o  solutions  during  150 
days.  The  quantities  are 
stated  in  cubic  centimeters 
and  the  gain  in  length  of 
thallus  in  per  cent.  I, 
NaCl  1000  +  CaCl2  10, 
gain  820.  2,  NaCl  1000 
+  KC1  22  +  CaCl3  10,  gain 
980.  3,  distilled  water, 
gain  1220.  4,  NaCl;  5, 
KC1;  6,  MgCl2;  gain  o. 


1907] 


OSTERHOUT— BALANCED  SOLUTIONS 


263 


The  gemmae  were  removed  from  the  cups  with  the  point  of  a 
knife  and  sprinkled  on  the  surface  of  the  solution,  care  being  taken 
to  exclude  particles  of  dust  and  dirt.  Each  tumbler  contained  2oocc 
of  solution  and  was  covered  with  a  glass  plate.  The  gemmae  may 
be  easily  removed  from  the  surface  of  the  solution  for  purposes  of 
observation  by  dipping  into  it  a  clean  slide  and  slowly  raising  it  at  an 
angle  so  as  to  take  up  the  material  upon  one  side  only.  The  material 
should  not  be  replaced  in  the  solution  unless  extreme  care  be  taken 
to  prevent  contamination. 

The  average  results  of  six  series  of  experiments  are  given  in  Tables 
IV  and  V,  and  illustrated  in  figs.  2  and  j. 


TABLE  IV,     LUNULARIA 

A  plus  sign  indicates  that  the  plants  were  alive  when  the  experiment  was  stopped. 
All  quantities  given  are  cubic  centimeters    of  3^/32  solutions. 


CULTURE  SOLUTION 


Dilute  sea  water;  total  salts 

0.6  per  cent 

Dilute  artificial  sea  water: 
1000  NaCl 
78  MgCl2 
38  MgS04 
22  KC1 
10  CaCl2 

Distilled  water. . .  . 
NaCl. .  . 
1000  NaCl 
10  CaCl2 

1000  NaCl 
22  KC1 
10  CaCl2 

1000  NaCl 
78  MgCl2 
10  CaCl2 

1000  NaCl 
78  MgCl2 
22  KC1 

1000  NaCl 
22  KC1 

1000  NaCl 
78  MgCl2 
MgCl2. 
KC1  .. 
CaCl2.. 


DURATION  OF  LIFE  IN  DAYS 


200  + 


200  + 


2OO  + 

4 


200  + 


2OO  + 


2 

12 
100 


264 


BOTANICAL  GAZETTE 


[OCTOBER 


TABLE  V.     LUNULARIA 
All  quantities  given  are  cubic  centimeters  of  3^/80  solutions 


( 

GROWTH  IN  150  DAY 

s 

CULTURE  SOLUTION 

Length  of  thallus 
in.  tnm. 

Per  cent,  increase  in 
length  of  thallus 

Aggregate  length 
of  rhizoids  per 
thallus  in  mm. 

Dilute  artificial  sea  water: 

1000  Nacl        \ 

78   MgCla 

38  MgSO4   )  . 

5.52 

1  2O4. 

1  68 

22    KC1 

J.  —  W-|. 

10  CaCla      ' 

Distilled  water    . 

5.60 

I22O 

180 

NaCl     

O  .  CO 

o 

o 

1000  NaCl       I 

d.   60 

82O 

i  id. 

10  CaCla      )  

*r  •  ww 

j.  j.£f. 

1000  NaCl       ) 

22    KC1             [• 

^  .  4.O 

080 

c 

10  CaCla      ; 

' 

1000  NaCl       ) 

78  MgCla     [  

5-50 

IOOO 

125 

10  CaCla      ) 

1000  NaCl       ) 

78  MgCl3     [  

0.54 

8 

0.3 

22    KC1             ) 

1000  NaCl       > 

22  KCI     r 

0.50 

o 

o 

1000  NaCl       ) 

O.  C2 

O  .  I 

78  MgCla     J  

MgCla  

0.50 

o 

o 

KCI  

0.50 

o 

o 

CaCla 

4ee 

810 

•  j  j 

' 

EQUISETUM 

The  spores  of  Equisetum  retain  their  vitality  for  only  a  few  days. 
The  fruiting  cones  were  brought  into  the  laboratory  and  allowed  to 
stand  for  a  day  or  two.  The  freshly  shed  spores  were  then  placed  on 
the  surfaces  of  solutions  in  covered  glass  dishes.  They  germinate 
rapidly  and  in  a  few  days  produce  prothallia  of  fair  size.  The  aver- 
age results  of  four  series  of  experiments  are  shown  in  Table  VI  and 
fig-4- 

FLOWERING   PLANTS 

The  most  extensive  series  of  experiments  was  made  with  wheat. 
The  variety  selected  is  known  as  Early  Genesee.  The  percentage 


1907] 


OSTERHOUT— BALANCED  SOLUTIONS 


265 


of  germination  is  very  high  and  the  growth  is  vigorous  from  the  start. 
The  plan  first  tried  was  that  of  carefully  placing  the  seeds  on  the 
surface  of  the  solutions  so  that  they  float.  This  worked  well  with 


O     Q     0 


FIG.  3. — Growth  of  gem- 
mae of  Lunularia  in  various 
3w/8o  solutions  during  150 
days.  The  quantities  are 
stated  in  cubic  centimeters, 
and  the  gain  in  length  of 
thallus  in  per  cent.  I,  CaCl2, 
gain  810.  2,  NaCl  1000 
+  MgCl2  78  +  CaCla  10, 
gain  1000.  3,  dilute  arti- 
ficial sea  water,  NaCl  =  ap. 
3w/8o,  gain  1204.  4, 
NaCl  1000  +  KC1  22, 
gain  o.  5,  NaCl  1000 
+  MgCl2  78,  gain  4.  6, 
NaCl  1000  +  MgCl2  78 
+  KC1  22,  gain  8. 


266 


BOTANICAL  GAZETTE 


[OCTOBER 


wheat  and  other  small  seeds  during  the  first  stages  of  germination; 
but  if  the  experiments  are  to  be  carried  beyond  this  stage,  the  seed- 
lings must  be  supported  so  that  the  leaves  do  not  come  into  contact 

with  the  solution.  After 
some  trials  the  following 
device  was  hit  upon  which 
answers  the  purpose 
admirably.  A  strip  of 
filter  paper  is  folded 
lengthwise  and  one  of 
the  folds  turned  back  as 
shown  in  fig.  5.  The 
seeds  are  placed  in  the 
trough  thus  formed  and 
the  whole  strip  is  then 
bent  into  a  circle  and 
placed  in  a  tumbler  previ- 
ously filled  with  solution. 
The  strip  should  be  of 
such  length  that  when 
placed  in  the  top  of  the 
tumbler  the  ends  just 
meet  and  so  form  a  stiff 
collar  which  just  fits 
inside  the  top  of  the 
tumblers  and  which  will 


FIG.  4. — Development  of  spores 
of  Equisetum  in  various  3^/160 
solutions  during  50  days.  Quanti- 
ties are  stated  in  cubic  centi- 
meters; the  gain  in  length  of 
thallus  exclusive  of  rhizoids  is 
stated  in  per  cent.  I,  distilled 


water,  gain  1760.  2,  dilute  arti- 
ficial sea  water,  NaCl  =  ap. 
3w/i6o,  gain  1500.  3,  NaCl 
1000  +  KC1  22  -f  Cada  10,  gain 
1500.  4,  NaCl  iooo+CaCl2  10, 
gain  980.  5,  CaCl2,  gain  700. 
6,  NaCl,  gain  o. 


not  slip  down.  A  large 
number  of  these  collars 
may  be  prepared,  filled 
with  seeds,  bent  into 
circles,  and  secured  by 
ordinary  paper-clips 

placed  on  the  overlapping  ends.     They  may  be  piled  in  trays  until 

wanted.     It  is  then  only  necessary  to  remove  the  clips  and  set  the 

collars  in  glasses  previously  filled  with  solutions. 

In  some  cases,  especially  where  larger  glasses  are  employed,  a 

strip  of  paper  of  double  thickness  may  be  used ;  this  makes  a  stiff er 


1907] 


OSTERHOUT— BALANCED  SOLUTIONS 


267 


collar.  It  is  then  advisable  to  perforate  the  bottom  of  the  seed  trough 
by  means  of  a  tracing  wheel  such  as  is  used  for  patterns.  This  allows 
the  roots  to  penetrate  the  paper  freely  and  without  delay. 

Care  should  be  taken  that  the  solution  does  not  cover  the  seeds. 
The  paper  must  be  spread  open  at  the  top  so  as  to  allow  the  air  to 
come  into  direct  contact  with  the  seeds.  The  micropyle  should  be  in 
contact  with  the  moist  filter  paper. 

Careful  experiments  were  made  to  determine  whether  the  filter 
paper  exerted  any  influence  on  the  solution  (by  absorption,  etc., 
or  by  concentration  of  the  solution  about  the  see'd  as  the  result  of 
evaporation)  which  might  affect  the  results,  but  no  such  influence 

TABLE  VI.     EQUISETUM 
All  quantities  given  are  cubic  centimeters  of  3^/160  solutions. 


GROWTH  IN  50  DAYS 

j 

CULTURE  SOLUTION 

length  of  thallus 
in  mm. 

Per  cent,  increase  in 
length  of  thallus, 
exclusive  of 
rhizoids 

Aggregate  length 
of  rhizoids  per 
thallus  in  mm. 

Dilute  artificial  sea  water: 

1000  NaCl 

78  MgCl2 

38  MgSO4       

0.8o 

I^OO 

8.1 

22    KC1 

o 

10  CaCl2 

Distilled  water 

o  .  93 

1760 

9.0 

NaCl  

O.OtC 

A  1  \J\4 

o 

y 

o 

1000  NaCl 

o 
o.  54 

980 

"v^ 

4.7 

10  CaCl2 

*T  *  / 

1000  NaCl       ) 

22    KC1              [•   

10  CaCl2      ) 

0.80 

1500 

5-2 

1000  NaCl       ) 

78  MgCl2     [  

o-93 

1760 

9.0 

10  CaCl2      ) 

1600  NaCl       ) 

78  MgCl        r 

0.07 

40 

O 

22    KC1     2        ) 

1000  NaCl       I 

o 

o 

22    KC1              J    • 

' 

1000  NaCl       I 

0.07 

40 

o 

78  MgCl2     )  

•    / 

MeCU 

o.oc 

o 

o 

KC1                      .    ... 

0 

o 

o 

CaCl2          

0.40 

700 

3  •  2 

/ 

268 


BOTANICAL  GAZETTE 


[OCTOBER 


could  be  detected.    The  solutions  were  renewed  from  time  to  time  and 
the  concentration  ascertained  by  occasional  titration. 

It  should  be  said  that  in  general  the  growth  of 
roots  (or  any  parts  in  direct  contact  with  the 
solution)  furnishes  a  much  better  criterion  of  the 
effect  of  solutions  than  the  aerial  portions  of  the 
plant.  In  certain  solutions  which  are  so  poisonous 
that  the  roots  cannot  develop,  the  leaves  may  grow 
fairly  well  for  a  time.  In  these  cases  the  poisonous 
solutes  are  apparently  filtered  out  by  the  tissues 
of  the  seed  as  the  solution  passes  through  them 
on  its  way  to  the  leaf.  For  this  reason  the  figures 
for  the  growth  of  roots  only  are  here  given.  The 
results  are  shown  in  Table  VII  and  figs.  6  and 
7,  which  give  the  average  of  five  series  of  ex- 
periments. Each  number  represents  average 
measurements  of  at  least  four  or  five  hundred 
seeds.  This  is  necessary  in  order  to  do  away  with 
the  individual  variation  so  common  in  seeds. 


FIG.  5. — Sectional 
view  of  wall  of  tumb- 
ler and  seed  sup- 
ported by  folded 
filter  paper;  p,  paper; 
s,  seed;  t,  tumbler; 
•w,  water  line. 


TABLE  VII.    WHEAT 
All  quantities  given  are  cubic  centimeters  of  3^/25  solutions. 


CULTURE  SOLUTION 


Dilute  artificial  sea  water: 
1000  NaCl 
78  MgCl, 
38  MgS04 
22  KC1 
io  CaCl2 

Distilled  water. . .  . 
NaCl... 
1000  NaCl 
io  CaCl2 

1000  NaCl 
22  KC1 
io  CaCl2  ' 

1000  NaCl 
78  MgCla 
io  CaCl2 
MgCl2.. 
KC1.... 
CmCl... 


GROWTH  IN  40  DAYS 


Aggregate  length  of  roots 
per  plant  in  mm. 


360 

59 

254 

324 

327 

68 

70 


1907] 


OSTERHOUT— BALANCED  SOLUTIONS 


269 


A  similar  though  less  extensive  series  of  experiments  was  carried 
out  with  flax,  alfalfa,  red-beet,  and  radish  seeds.  Another  series 
was  made  by  placing 
pieces  of  the  fresh- 
water aquatics, 
Zannichellia  and 
Potamogeton,  in 
solutions,  or  in  the 
case  of  Lemna,  by 
allowing  the  plants 
to  float  on  the  sur- 
face. The  results 
in  all  these  cases 
were  similar  to  those 
given  above. 

It  is  thought 
desirable  to  see  how 
cuttings  would 
behave  under  simi- 
lar treatment.  Cut- 
tings (about  nine 

inches  in  length)   of  Tradescantia   and  Tropaeolum  were  placed 
upright  in  bottles,  the  lower  three  inches  of  the  plant  being  submerged 

TABLE  VIII.     CUTTINGS 

All  quantities  given  are  cubic  centimeters  of  3^/32  solutions. 


FIG.  6. — Growth  of  roots  of  wheat  in  various  3^/25 
solutions.  Quantities  are  stated  in  cubic  centimeters, 
and  the  aggregate  length  of  roots  in  millimeters.  I,  NaCl 
1000  +  KC1  22  +  CaCl2  10,  length  324.  2,  NaCl  1000 
+  CaCl2  10,  length  254.  3,  CaCl2,  length  70.  4,  NaCl, 
length  59. 


GROWTH  IN  10  DAYS 


UULTURE   bOLUTION 

Tradescantia 

Tropaeolum 

Dilute  artificial  sea  water: 
1000  NaCl 
78  MgCl2 
?8  MgSO4      . 

Long  roots 

Long  roots 

22    KC1 

10  CaCl2 
Distilled  water 

Very  long  roots 

Very  long  roots 

NaCl  

No  roots 

No  roots 

1000  NaCl       I 

Short  roots 

Short  roots 

ioCad2        )  

1000  NaCl      ) 
22  KC1        [  

Medium  length  roots 

Medium  length  roots 

10  CaCl2      ) 

270 


BOTANICAL  GAZETTE 


[OCTOBER 


in  the  solution.  Absorbent  cotton  was  packed  in  the  neck  of  the 
bottle  to  exclude  dirt  and  hinder  evaporation.  The  results  were 
similar  to  those  described  above,  as  will  be  seen  from  Table  VIII. 

Finally  the  question  was  raised  whether 
the  tissues  of  the  stem  and  leaf,  if  brought 
into  direct  contact  with  the  solution,  would 
behave  like  the  root.  To 
answer  this,  sections  of 
considerable  (but  uni- 
form) thickness  were  cut 
with  a  microtome  and 
placed  in  the  solutions. 
The  results  appear  in 
Table  IX. 

The  results  described 
in  this  paper  are  in  all 
essentials  in  striking 
agreement  with  those 
obtained  from  the  study 
of  marine  plants,  as  well 
as  from  the  study  of 
marine  and  fresh-water 
animals  as  referred  to  in 
the  first  part  of  this 
paper.  This  agreement 
shows  that  the  principle 
of  balanced  solutions  is 
of  general  validity.3  The 
application  of  this  prin- 
ciple to  soil  and  river  water4  and  to  nutrient  solutions,  I  hope  to  take 
up  in  a  subsequent  paper. 

3  LOEW  and  his  pupils  have  shown  that  calcium  antagonizes  magnesium  (cf. 
Bull.  No.  18,  Div.  Veg.  Phys.  and  Path.  U.  S.  Dept.  Agric.  1899).     See  also  the  antago- 
nistic effects  noted  by  KEARNEY  and  CAMERON  (Report  No.  71,  U.  S.  Dept.  Agric. 
1902)  in  their  studies  on  the  salts  of  alkali  soils.     The  method  employed  by  them 
(observation  of  the  root-tip  only)  is  so  different  from  mine  that  I  have  not  attempted 
to  compare  the  results. 

4  In  the  first  part  of  this  paper  I  have  referred  to  the  composition  of  tap  water, 
but  it  seems  advisable  to  defer  the  discussion  of  this  point. 


FIG.  7. — Growth  of  roots  of  wheat  for  40  days. 
I,  in  dilute  artificial  sea  water  (NaCl  =  ap.  3^/25), 
aggregate  length  of  roots  360""".  2,  in  distilled 
water,  aggregate  length  of  roots  74omm. 


1907] 


OSTERHOUT— BALANCED  SOLUTIONS 


271 


TABLE  IX.     SECTIONS 
All  quantities  given  are  cubic  centimeters  of  3^/32  solutions. 


DURATION  OF  LIFE  IN  DAYS 

CULTURE  SOLUTION 

Red  beet: 
Cross-sections 
of  root 

Mesembry- 
anthemum  : 
Cross-sections 
of  leaf 

Tradescantia  : 
Cross-sections 
of  stem 

Tropaeolum: 
Cross-sections 
.aLleaf 

Dilute  artificial  sea 

water: 

1000  NaCl 

. 

78  MgCl3 

38  MgSO,, 

•2C 

16 

10 

2C 

O       j-»j-£o\-^4 
22    KC1 

j  o 

y 

•j 

10  CaCl2 

Distilled  water 

•7C 

18 

2O 

•72 

NaCl 

•JJ 

18 

12 

O 
14. 

1000  NaCl 
10  CaCla 

! 

" 

25 

12 

18 

xiT 

20 

1000  NaCl 

I 

22    KC1 

10 

je 

10 

22 

10  CaCl2     , 

i 

o 

j 

y 

For  the  sake  of  clearness  it  seems  desirable  to  call  attention  to  the 
distinction  between  balanced  solutions  and  ordinary  nutrient  solu- 
tions. A  nutrient  solution  may  be  used  in  such  dilute  form  that  none 
of  its  components  could  exert  any  toxic  action  even  if  the  other  con- 
stituents were  removed.  In  this  case  there  are  no  poisonous  effects 
to  be  inhibited  and  consequently  no  balancing  of  the  solution  is 
required.  Our  only  concern  is  to  supply  all  the  substances  needed 
for  nutrition,  irrespective  of  any  balancing  action,  and  so  form  a 
complete  nutrient  solution. 

If  we  increase  the  concentration  of  this  solution,  however,  we  soon 
reach  the  point  where  some  or  all  of  the  components  begin  to  exert 
their  individual  toxic  effects,  whereupon  it  may  become  necessary 
to  inhibit  these  effects  by  proper  adjustment  of  the  relative  propor- 
tions of  the  substances  present  or  by  the  addition  of  other  substances. 
The  substances  added  to  produce  a  balance  do  not  necessarily  have  a 
nutritive  value.  For  example,  LOEBS  was  able  to  balance  certain 
solutions  by  adding  zinc,  cobalt,  aluminum,  etc.6 

s  Am.  Jour.  Physiology  6:411-433.   1902. 

6  To  make  clear  this  distinction  between  balanced  and  nutrient  solutions  is  more 
necessary,  since  LOEW  and  Aso  (Bull.  Coll.  Agr.  Tokyo  Imp.  University  7:395.  1907) 
confuse  the  two  kinds  of  solutions.  Their  criticisms  are  wholly  based  on  this  mis- 
conception and  do  not  affect  the  matter  as  I  have  presented  it.  The  distinction 
between  nutrient  and  balanced  solutions  is  due  to  LOEB,  who  has  explained  it  clearly 
in  his  Dynamik  der  Lebenserscheinungen  115-120. 


272  BOTANICAL  GAZETTE  [OCTOBER 

In  general  we  may  know  when  the  solution  is  properly  balanced 
by  comparing  its  effects  with  those  of  pure1  distilled  water.  In  a 
properly  balanced  solution  we  expect  the  organism  to  live  approxi- 
mately as  long  as  in  distilled  water,  and  while  it  will  not  grow  so  fast 
(on  account  of  the  osmotic  pressure),  the  ultimate  development  reached 
should  be  comparable  with  that  attained  in  distilled  water.8 

Why  all  these  effects  are  so,  we  are  not  at  present  prepared  to  say 
in  detail.  LOEB  has  gone  farther  than  any  other  in  the  explanation 
of  these  phenomena,  referring  them  to  the  effects,  of  salts  and  ions  on 
proteids9.  According  to  his  conception  any  metal  must  be  poisonous 
when  it  alone  is  present  in  the  solution,  for  it  will  enter  the  proteids 
and 'drive  out  other  metals  in  accordance  with  the  law  of  mass  action. 
This  will  of  course  alter  the  properties  of  the  proteids  and  so  cause 
disturbances  in  function.  The  only  way  to  prevent  this  is  to  main- 
tain a  proper  balance  between  the  various  metals  in  the  solution. 
It  may  be  pointed  out  that  an  analogy  exists  between  the  effects 
described  here  and  various  reactions  in  which  proteids  are  in- 
volved. Antagonism  between  Na  and  Ca,  for  example,  is  seen  in 
the  clotting  of  blood,  which  is  hindered  by  Na  and  favored  by  Ca. 

The  thing  of  chief  importance  is  the  agreement  in  behavior  of  such 
a  great  diversity  of  plants  with  the  fresh-water  and  marine  animals 
already  studied.  Thereby  is  brought  to  light  a  new  point  of  similarity 
between  animals  and  plants  which  is  fundamental  in  character  and 
which  must  be  taken  into  consideration  in  attempting  to  formulate 
a  theory  of  living  matter. 
UNIVERSITY  OF  CALIFORNIA 

7  Water  twice  distilled  from  glass,  the  first  third  of  the  distillate  being  rejected 
is  usually  regarded  as  pure.     But  such  water  may  be  quite  poisonous  if  any  part  of 
the  apparatus,  including  stoppers,  be  new.     The  longer  the  apparatus  is  used  the  less 
poisonous  the  water  becomes,  until  it  finally  ceases  to  be  toxic. 

8  Higher  concentrations  excepted. 

9  See  references  in  the  first  part  of  this  paper,  BOT.  GAZ.  42: 134.   1906 


14  DAY  USE 

RETURN  TO  DESK  FROM  WHICH  BORROWED 


ffS&y 

This  book  is  due  on  the  last  date  stamped  below,  or 

on  the  date  to  which  renewed. 
Renewed  books  are  subject  to  immediate  recall. 


1SS3 


LD  21-50m-12,'61 
(C4796slO)476 


General  Library 

University  of  California 

Berkeley 


